Interpretive Summary: Avian influenza virus is normally found in wild birds, but they virus can be transmitted to poultry where it can become an established infection. Avian influenza virus can cause a mild disease, called low pathogenic avian influenza, or a severe disease, called highly pathogenic avian influenza. Although there are 16 uniques strains or subtypes of avian influenza (H1-H16), only the H5 or H7 subtypes may cause the highly pathogenic form of the virus. Fortunately most H5 and H7 strains are low pathogenic, but potentially they can mutate to the highly pathogenic form. We know one critical factor for this increase in virulence in chickens is at the hemagglutinin cleavage site. We examined some H7 low pathogenic avian influenza viruses found in the U.S. to try and determine what changes would be necessary for the viruses to become highly pathogenic. Our studies showed that an insertion of new RNA at the cleavage site was necessary to become highly pathogenic, and simple substitution changes did not change the virulence of the virus. This information is important in helping us understand how avian influenza viruses increase in virulence, and can help us identify dangerous viruses in the future.

Technical Abstract:
An H7N2 subtype avian influenza virus (AIV) first appeared in live-bird marketing system (LBMS) in the Northeastern United States in 1994. Since then this lineage of virus has become the predominant subtype of AIV isolated from the LBMS, and has been linked to several costly commercial poultry outbreaks. Concern for this low pathogenicity isolate mutating to the highly pathogenic form has remained high because of the increasing number of basic amino acids at the hemagglutinin (HA) cleavage site, which is known to be associated with increased pathogenicity of AIV. To address the risk of low pathogenic LBMS-lineage H7N2 virus mutating to the highly pathogenic form of the virus, we generated a series of mutant viruses that have changes in the sequence at the HA cleavage site by using plasmid-based reverse genetics. We confirmed that a conserved proline at –5 position from the HA cleavage site could be changed to basic amino acids, producing a virus with five basic amino acids in a row at the cleavage site, but with no increase in virulence. Increased virulence was only observed when additional basic amino acids were inserted. We also observed that the virus preferred the arginine instead of lysine at the –4 position from the cleavage site to manifest increased virulence both in vitro and in vivo. Using helper virus-based reverse genetics, where only one transcription plasmid expressing a mutated HA vRNA is used, we identified specific HA cleavage site sequences that were preferentially incorporated into the low pathogenic wild type virus. The resultant reassortant viruses were highly pathogenic in chickens. This study provides additional evidence that H7 avian influenza viruses require an insertional event to become highly pathogenic, as compared to H5 viruses that can become highly pathogenic strictly by mutation or by insertions.